Regulation of Steroid Hormone Production by Inter-Organelle Substrate Exchange

描述(申请人提供)：类固醇激素，如皮质醇，是多种生物过程的关键调节器，通过与核受体转录因子超家族成员结合而引起生理变化。由于糖皮质激素控制许多基因的表达，因此使用多种机制来确保严格控制激素的产生。我们的初步发现为促肾上腺皮质激素(ACTH)刺激的微管依赖的线粒体运输在维持最佳糖皮质激素输出中的作用提供了证据。我们还鉴定了几种蛋白质，包括GTP酶RhoA和Rho效应器透明1(DIAPH1)，它们促进了这一运动。ACTH信号控制DIAPH1与RhoA和其他几个结合伙伴相互作用的能力。然而，这些相互作用的功能意义还没有完全阐明，我们也没有定义ACTH刺激的线粒体运动是否通过促进内质网和线粒体之间底物的传递来促进皮质醇的产生。我们假设ACTH刺激的线粒体转运速率的增加是细胞器间底物交换和糖皮质激素生物合成所必需的。为了验证这一假设，我们将定义ACTH信号控制DIAPH1复合体组装的机制(特定目标1)。我们已经建立了一个稳定的细胞系，它表达针对DIAPH1的shRNA，以确定该蛋白在线粒体运动和激素产生中的作用。我们还将评估DIAPH1与几个结合伙伴之间的相互作用的功能意义，这些结合伙伴包括微管蛋白、动蛋白和氧固醇结合蛋白相关蛋白2(ORP2)。还将进行研究，以确定磷酸化在控制DIAPH1功能中的作用(特定目标2)。我们发现ACTH信号刺激DIAPH1的磷酸化。这种翻译后修饰对DIAPH1功能的作用将使用磷酸化突变体和磷酸化特异性抗体来检验。最后，我们将确定11-脱氧皮质醇在内质网和线粒体之间的转运机制(具体目标3)。我们发现ORP2与DIAPH1共同纯化，并假设这种脂结合蛋白可能促进底物在内质网和线粒体之间的转移。我们将确定ORP2在皮质醇生物合成中的作用，并确定内质网和线粒体之间的融合是否在细胞器间底物输送中发挥作用。从这些研究中获得的知识将提供关于肾上腺皮质类固醇激素合成的一个基本上未被探索的方面的有价值的信息。我们的发现也可能为在其他代谢过程中底物在内质网和线粒体之间转移的机制提供洞察力。

DESCRIPTION (provided by applicant): Steroid hormones such as cortisol are key regulators of a diverse array of biological processes that evoke physiological changes by binding to members of the nuclear receptor super family of transcription factors. Because glucocorticoids control the expression of numerous genes, multiple mechanisms are used to ensure tight control over hormone production. Our preliminary findings provide evidence for the role of adrenocorticotropin (ACTH)-stimulated, microtubule-dependent mitochondrial trafficking in maintaining optimal glucocorticoid output. We have also identified several proteins, including the GTPase RhoA and the Rho effector diaphanous 1 (DIAPH1), that act to facilitate this movement. ACTH signaling controls the ability of DIAPH1 to interact with RhoA and several other binding partners. However, the functional significance of these interactions has not fully been elucidated, nor have we defined if ACTH-stimulated mitochondrial movement enables cortisol production by facilitating the delivery of substrate between the ER and mitochondria. We hypothesize that ACTH-stimulated increases in the rate of mitochondrial trafficking are required for inter- organelle substrate exchange and glucocorticoid biosynthesis. To test this hypothesis we will define the mechanism by which ACTH signaling controls DIAPH1 complex assembly (Specific Aim 1). We have generated a stable cell line that expresses shRNA against DIAPH1 to define the role of the protein in mitochondrial movement and hormone production. We will also assess the functional significance of the interactions between DIAPH1 and several binding partners, including tubulin, kinesin, and the oxysterol- binding-protein related protein 2 (ORP2). Studies will also be performed to define the role of phosphorylation in controlling DIAPH1 function (Specific Aim 2). We have found that ACTH signaling stimulates the phosphorylaton of DIAPH1. The role of this post-translational modification on DIAPH1 function will be examined using phospho-mutants and a phospho-specific antibody. Finally, we will determine the mechanism by which 11-deoxycortisol is transported between the ER and mitochondria (Specific Aim 3). We have found that ORP2 co-purifies with DIAPH1 and hypothesize that this lipid binding protein may facilitate substrate transfer between the ER and mitochondria. We will characterize the role of ORP2 in cortisol biosynthesis and also determine if fusion between ER and mitochondria play a role in inter-organelle substrate delivery. The knowledge gained from these studies will provide valuable information about a largely unexplored facet of steroid hormone synthesis in the adrenal cortex. Our findings may also provide insight into the mechanism by which substrates are transferred between the ER and mitochondria in other metabolic processes.